Method of detecting nucleotide polymorphism

ABSTRACT

A Nucleotide useful for detecting a base substitution in a gene, a method for detecting a base substitution in a gene using said Nucleotide, and a kit for the same.

TECHNICAL FIELD

[0001] The present invention relates to a Nucleotide (an oligonucleotideto be used for the method of the present invention) useful for detectinga base substitution in a gene, a method for detecting a basesubstitution in a gene using said Nucleotide, and a kit for the same.

BACKGROUND ART

[0002] It is known that genetic codes contained in genomes of organismindividuals belonging to the same species are not identical each other,and there are differences in base sequences called polymorphisms. Onesin which one to tens of base(s) is (are) deleted or inserted, ones inwhich a specific base sequence is duplicated and the like are known aspolymorphisms. One in which one base is replaced by another base iscalled a single nucleotide polymorphism (SNP).

[0003] It is said that single nucleotide polymorphisms exist at a rateof about one per hundreds to one thousand bases. Accordingly, the numberof SNPs present on a human genome is estimated to be three to tenmillion. Attentions are paid to SNPs as indexes for searching for genesrelated to diseases, or for having information about differences insusceptibilities to diseases or sensitivities to drugs (actions or sideeffects). Methods for detecting SNPs are under study.

[0004] Conventional means for detecting SNPs are generally classifiedinto ones based on hybridization, ones based on primer extension andones utilizing substrate specificities of enzymes.

[0005] The presence of a base substitution is detected by means ofhybridization of a probe to a nucleic acid sample in a hybridizationmethod. According to the method, it is necessary to determine a probeand hybridization conditions so that hybridization is influenced by adifference in one base. Therefore, it is difficult to establish a highlyreproducible detection system.

[0006] A method for detecting a mutation using a cycle probe reaction asdescribed in U.S. Pat. No. 5,660,988 is exemplified. A nucleic acidprobe having a readily cleavable binding is hybridized to a nucleic acidmolecule of interest in the method. If the nucleic acid molecule ofinterest does not have a base substitution, the probe is cleaved,whereas if the nucleic acid molecule has a base substitution, the probeis not cleaved. A base substitution is then detected by detecting andquantifying the degree of generation of a fragment released from thecleaved probe. However, if a trace amount of a target nucleic acid is tobe detected according to this method, there may be a considerable timelag until reaching a level at which one can detect a cleavage productfrom the probe because the amount of the cleavage product is small.

[0007] A method for detecting a mutation using the TaqMan method asdescribed in U.S. Pat. Nos. 5,210,015 and 5,487,972 exemplifies anothermethod. A TaqMan probe to which a fluorescent dye and a quencher areattached is used in this method. Two probes (one containing a basesubstitution and the other containing no base substitution) are used asthe TaqMan probes. The probe is hybridized to a nucleic acid molecule ofinterest, and a primer is extended from the upstream. The probe iscleaved due to a 5′→3′ exonuclease activity of a DNA polymerase only ifthe nucleic acid molecule of interest does not contain a basesubstitution. A base substitution is then detected by detecting emittedfluorescence. However, the method has problems because the methodrequires a polymerase having a 5′→3′ exonuclease activity, a PCR using alabeled nucleotide blocked at the 3′-terminus and a strict temperatureadjustment, and it requires a long time for detection.

[0008] Methods in which an enzyme is utilized include methods in which aDNA polymerase is used. Such methods are further classified into threegroups as follows: (1) methods in which a base substitution is detectedbased on the presence of a primer extension reaction using a primer ofwhich the 3′-terminus anneals to a base portion for which a basesubstitution is to be detected as described in U.S. Pat. No. 5,137,806;(2) methods in which a base substitution is detected based on thepresence of a primer extension reaction using a primer in which the basesubstitution site to be detected is located at the second nucleotidefrom the 3′-terminus as described in WO 01/42498; and (3) method inwhich the presence of a mutation at the site of interest and the base atthe site are determined by distinguishing a base incorporated into aprimer using a primer of which the 3′-terminus anneals to a base 3′adjacent to the base for which a base substitution is to be detected.

[0009] Methods in which a DNA ligase is used are known. According to themethod, a base substitution is detected based on the presence ofligation of a probe to an adjacent probe. The terminal portion of theprobe corresponds to the base portion for which a base substitution isto be detected.

[0010] A method in which a DNA polymerase or a DNA ligase is used maynot be able to exactly detect a mismatch between a primer (or a probe)and a target nucleic acid due to a base substitution. Specifically, suchan enzyme may initiate an enzymatic reaction even if the primer or theprobe has a mismatch, providing erroneous results.

[0011] Because of a possible false positive due to an erroneousannealing between a target nucleic acid and a primer or an error made bya ligase or a polymerase to be used, it is necessary to control thereaction conditions (in particular, the reaction temperature) and thelike very strictly, and there is a problem concerning thereproducibility.

[0012] Lastly, methods in which an enzyme having an activity ofrecognizing and cleaving a specific structure in a double-strandednucleic acid is utilized such as the invader method as described in U.S.Pat. No. 5,846,717 are included. A cleavase is known as such an enzyme.It is possible to detect a base substitution by examining cleavage of aprobe. The probe is designed such that it forms a structure recognizedby the enzyme if a base substitution is present (or absent). However,such a method in which an enzyme having an activity of recognizing andcleaving a specific structure in a double-stranded nucleic acid is usedhas a problem concerning its sensitivity. Specifically, a signalsufficient for detection of a base substitution cannot be obtained froma trace amount of a nucleic acid sample since one signal is generatedfrom a distinct target nucleic acid molecule according to the method. Itis naturally possible to enhance the signal by repeating the probecleavage reaction, although it is necessary to amplify a target nucleicacid beforehand in order to obtain an intense signal. Thus, if a traceamount of a target nucleic acid is to be detected according to thismethod, there may be a considerable time lag until reaching a level atwhich one can detect a cleavage product from the probe because theamount of the cleavage product is small.

[0013] Since the methods have several problems as described above, amethod that can be used to exactly detect a base substitution has beendesired.

OBJECTS OF INVENTION

[0014] The main object of the present invention is to solve theabove-mentioned problems and to provide a means for detecting a basesubstitution (e.g., an SNP) exactly with excellent reproducibility evenif a trace amount of a nucleic acid sample is used.

SUMMARY OF INVENTION

[0015] In order to solve the problems as described above, a method thatcan be used to exactly detect a base substitution and obtain results asintense signals is desired.

[0016] The present inventors have prepared a Nucleotide. The Nucleotideis capable of annealing to a target nucleic acid for which a basesubstitution is to be detected. A DNA extension reaction from its3′-terminus by a DNA polymerase is not initiated if the Nucleotide is inan intact state. Cleavage of the Nucleotide by a nuclease is influencedby the base sequence of the annealed template strand. Furthermore, thepresent inventors have established a method that can be used to detect abase substitution in a target nucleic acid exactly with high sensitivityusing the Nucleotide. Thus, the present invention has been completed.

[0017] The present invention is outlined as follows. The first aspect ofthe present invention relates to a method for detecting the presence ofa base substitution at a specific base in a target nucleic acid, themethod comprising:

[0018] (1) mixing a sample containing a target nucleic acid with aNucleotide, wherein the Nucleotide

[0019] (A) is modified at the 3′-terminus such that extension from theterminus by a DNA polymerase does not occur;

[0020] (B) has a base sequence capable of annealing to a regioncontaining a specific base in the target nucleic acid; and

[0021] (C) contains a sequence in which if there is a mismatch betweenthe specific base and a base corresponding to the specific base in theNucleotide in a complex composed of the Nucleotide and the targetnucleic acid, the Nucleotide is not cleaved with a nuclease, and ifthere is no mismatch between the specific base and a base correspondingto the specific base in the Nucleotide, the Nucleotide is cleaved with anuclease to generate a new 3′-terminus;

[0022] (2) treating the mixture with the nuclease and the DNApolymerase; and

[0023] (3) detecting the presence of cleavage of the Nucleotide with thenuclease.

[0024] The following methods exemplify the method for detecting a basesubstitution of the first aspect: a method wherein the nuclease is aribonuclease H, and the Nucleotide contains a ribonucleotide in theregion containing the base corresponding to the specific base; and amethod wherein the nuclease is a restriction enzyme, and the Nucleotidecontains a recognition sequence for the restriction enzyme in the regioncontaining the base corresponding to the specific base.

[0025] The second aspect of the present invention relates to a methodfor detecting a base substitution in a target nucleic acid, the methodcomprising:

[0026] (1) mixing a sample containing a target nucleic acid with aNucleotide, wherein the Nucleotide

[0027] (A) is modified at the 3′-terminus such that extension from theterminus by a DNA polymerase does not occur;

[0028] (B) has a base sequence capable of annealing to a regioncontaining a specific base in the target nucleic acid; and

[0029] (C) contains a sequence in which if there is no mismatch betweenthe specific base and a base corresponding to the specific base in theNucleotide in a complex composed of the Nucleotide and the targetnucleic acid, the Nucleotide is not cleaved with a nuclease, and ifthere is a mismatch between the specific base and a base correspondingto the specific base in the Nucleotide, the Nucleotide is cleaved with anuclease to generate a new 3′-terminus;

[0030] (2) treating the mixture with the nuclease and the DNApolymerase; and

[0031] (3) detecting the presence of cleavage of the Nucleotide with thenuclease.

[0032] A method wherein the nuclease is a mismatch-specific nucleaseexemplifies the detection method of the second aspect.

[0033] The Nucleotide used in the detection method of the first orsecond aspect may have a sequence in which if there is no basesubstitution in the target nucleic acid, a mismatch is not generated inthe complex composed of the Nucleotide and the target nucleic acid, orit may have a sequence in which if there is a base substitution in thetarget nucleic acid, a mismatch is not generated in the complex composedof the Nucleotide and the target nucleic acid.

[0034] The following methods exemplify embodiments of the first orsecond aspect: a method wherein the presence of a base substitution isdetermined based on the presence of an extension product generated bythe action of the DNA polymerase; and a method wherein the presence of abase substitution is determined based on the presence of a fragment of a3′ portion released from the Nucleotide generated by the action of thenuclease. Furthermore, an extension product or a fragment of a 3′portion of the Nucleotide can be detected utilizing a label attached tothe Nucleotide. A fluorescent substance may be used as the label.Furthermore, a Nucleotide to which a fluorescent substance and asubstance capable of quenching fluorescence are attached, wherein thefluorescence is emitted upon cleavage by the nuclease or DNA extensionsubsequent to the cleavage may be used. In the embodiment in which thefluorescence labeled Nucleotide is used, a fluorescence polarizationmethod may be utilized for detection.

[0035] In regard to the Nucleotide used in the method for detecting abase substitution of the first or second aspect, the modification of theNucleotide at the 3′-terminus is exemplified by modification of thehydroxyl group at the 3-position of ribose. The Nucleotide used in themethod for detecting a base substitution of the present invention maycontain a nucleotide analog and/or a modified nucleotide. Although it isnot intended to limit the present invention, for example, adeoxyriboinosine nucleotide, a deoxyribouracil nucleotide or the likemay be preferably used as a nucleotide analog, and an (α-S)ribonucleotide may be preferably used as a modified ribonucleotide.Furthermore, the method of the first or second aspect may furthercomprise a step of nucleic acid amplification in which an extensionproduct generated by the action of the DNA polymerase is used as atemplate.

[0036] The third aspect of the present invention relates to a method foranalyzing a genotype of an allele, the method comprising detecting abase substitution according to the method of the first or second aspect.

[0037] The fourth aspect of the present invention relates to aNucleotide used for detecting a base substitution at a specific base ina target nucleic acid, which

[0038] (A) is modified at the 3′-terminus such that extension from theterminus by a DNA polymerase does not occur;

[0039] (B) has a base sequence capable of annealing to a regioncontaining a specific base in the target nucleic acid; and

[0040] (C) contains a sequence in which if there is a mismatch betweenthe specific base and a base corresponding to the specific base in theNucleotide in a complex composed of the Nucleotide and the targetnucleic acid, the Nucleotide is not cleaved with a nuclease, and ifthere is no mismatch between the specific base and a base correspondingto the specific base in the Nucleotide, the Nucleotide is cleaved with anuclease to generate a new 3′-terminus.

[0041] The following Nucleotides exemplify the Nucleotide of the fourthaspect: a Nucleotide which contains a ribonucleotide in the regioncontaining the base corresponding to the specific base in the targetnucleic acid, wherein if there is no mismatch between the specific baseand the base corresponding to the specific base in the Nucleotide in acomplex composed of the Nucleotide and the target nucleic acid, theNucleotide is cleaved with a ribonuclease H; and a Nucleotide whichcontains a recognition sequence for a restriction enzyme in the regioncontaining the base corresponding to the specific base in the targetnucleic acid, wherein if there is no mismatch between the specific baseand the base corresponding to the specific base in the Nucleotide in acomplex composed of the Nucleotide and the target nucleic acid, theNucleotide is cleaved with the restriction enzyme.

[0042] The fifth aspect of the present invention relates to a Nucleotideused for detecting a base substitution at a specific base in a targetnucleic acid, which

[0043] (A) is modified at the 3′-terminus such that extension from theterminus by a DNA polymerase does not occur;

[0044] (B) has a base sequence capable of annealing to a regioncontaining a specific base in the target nucleic acid; and

[0045] (C) contains a sequence in which if there is no mismatch betweenthe specific base and a base corresponding to the specific base in theNucleotide in a complex composed of the Nucleotide and the targetnucleic acid, the Nucleotide is not cleaved with a nuclease, and ifthere is a mismatch between the specific base and a base correspondingto the specific base in the Nucleotide, the Nucleotide is cleaved with anuclease to generate a new 3′-terminus.

[0046] A Nucleotide wherein if there is a mismatch between theNucleotide and the target nucleic acid in a complex composed of theNucleotide and the target nucleic acid, the Nucleotide is cleaved with amismatch-specific nuclease exemplifies the Nucleotide of the fifthaspect.

[0047] The Nucleotide of the fourth or fifth aspect may have a sequencein which if there is no base substitution in the target nucleic acid, amismatch is not generated in the complex composed of the Nucleotide andthe target nucleic acid, or it may have a sequence in which if there isa base substitution in the target nucleic acid, a mismatch is notgenerated in the complex composed of the Nucleotide and the targetnucleic acid.

[0048] The Nucleotide of the fourth or fifth aspect may have a labeledcompound being attached. The position may be in a portion 3′ or 5′ tothe cleavage site for the nuclease. For example, a fluorescent substancemay be used as the labeled compound. By further attaching a substancecapable of quenching fluorescence, a Nucleotide from which thefluorescence is emitted upon cleavage by the nuclease or DNA extensionsubsequent to the cleavage may be prepared.

[0049] In regard to the Nucleotide of the fourth or fifth aspect, themodification of the Nucleotide at the 3′-terminus is exemplified bymodification of the hydroxyl group at the 3-position of ribose. TheNucleotide of the present invention may contain a nucleotide analogand/or a modified nucleotide. Although it is not intended to limit thepresent invention, for example, a deoxyriboinosine nucleotide, adeoxyribouracil nucleotide or the like may be preferably used as thenucleotide analog, and an (α-S) ribonucleotide may be preferably used asthe modified ribonucleotide.

[0050] The sixth aspect of the present invention relates to a kit usedfor detecting a base substitution in a target nucleic acid, whichcontains the Nucleotide of the fourth or fifth aspect.

[0051] The following kits exemplify the kit of the sixth aspect: a kitwhich contains a nuclease and/or a DNA polymerase; a kit which furthercontains a reagent for detecting the presence of DNA extension; and akit which further contains a reagent for carrying out a nucleic acidamplification method.

BRIEF DESCRIPTION OF DRAWINGS

[0052]FIG. 1 illustrates results for detection of a base substitution ina human gene according to the method for detecting a base substitutionof the present invention.

[0053]FIG. 2 illustrates results for detection of a base substitution ina human gene according to the method for detecting a base substitutionof the present invention.

[0054]FIG. 3 illustrates results for detection of a base substitution ina human gene according to the method for detecting a base substitutionof the present invention.

[0055]FIG. 4 illustrates results for detection of a base substitution ina human gene according to the method for detecting a base substitutionof the present invention.

[0056]FIG. 5 is a graph that illustrates results for detection of a basesubstitution in a human gene according to the method for detecting abase substitution of the present invention.

[0057]FIG. 6 illustrates results for detection of a base substitution ina human gene according to the method for detecting a base substitutionof the present invention.

[0058]FIG. 7 illustrates results for detection of a base substitution ina human gene according to the method for detecting a base substitutionof the present invention.

[0059]FIG. 8 illustrates results for detection of a base substitution ina human gene according to the method for detecting a base substitutionof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0060] As used herein, “a base substitution” refers to replacement of abase at a specific site in a nucleic acid by another base. The basesubstitution results in a difference in genetic information amongorganism individuals. The difference in genetic information is called apolymorphism or a variation. The base substitutions as used hereininclude base substitutions in polymorphisms and variations. The basesubstitutions also include base substitutions artificially introducedinto nucleic acids.

[0061] There is no specific limitation concerning the number ofsubstituted bases in the base substitution. There may be one or moresubstitutions.

[0062] The present invention is particularly suitable for detection of agenome polymorphism or a variation, in particular, a single nucleotidepolymorphism (SNP) in a gene.

[0063] The present invention is described in detail below.

[0064] (1) The Nucleotide of the Present Invention

[0065] The Nucleotide of the present invention has a base sequencecapable of annealing to a region containing a site in a target nucleicacid for which a base substitution is to be detected. The Nucleotidedoes not serve as a primer for DNA extension by a DNA polymerase if itis in an intact state, and it can serve as a primer only if it iscleaved by a nuclease. There is no specific limitation concerning thelength of the Nucleotide as long as it has the properties as describedabove. Both an oligonucleotide and a polynucleotide can be usedaccording to the present invention. An oligonucleotide of usually 8 to50 bases, preferably 10 to 40 bases, more preferably 12 to 30 bases isused as the Nucleotide of the present invention.

[0066] The Nucleotide of the present invention is usually anoligonucleotide containing deoxyribonucleotides. Optionally, it maycontain a ribonucleotide, or an analog or a derivative (modification) ofa nucleotide. For example, a nucleotide analog having a base such asinosine or 7-deazaguanine as its base moiety or a nucleotide analoghaving a ribose derivative can be used as a nucleotide analog. Examplesof modified nucleotides include an (α-S) nucleotide in which the oxygenatom attached to the phosphate group is replaced by a sulfur atom, and anucleotide to which a labeled compound is attached. Furthermore, theNucleotide of the present invention may contain a peptide nucleic acid(PNA) as described in Nature, 365:566-568 (1993). Although it is notintended to limit the present invention, the nucleotide analog orderivative is preferably incorporated at a site at which theincorporation does not influence the action of a nuclease to be used.Incorporation of a nucleotide analog into the Nucleotide of the presentinvention is effective in view of suppression of higher order structureformation of the Nucleotide itself and stabilization of annealing of theNucleotide to a target nucleic acid. Thus, the Nucleotide may contain anucleotide analog and/or a modified nucleotide as long as the functionas the Nucleotide that can be used in the method for detecting a basesubstitution of the present invention is retained.

[0067] The Nucleotide used according to the present invention has thefollowing properties for detection of a base substitution at a specificbase in a target nucleic acid:

[0068] (A) being modified at the 3′-terminus such that extension fromthe terminus by a DNA polymerase does not occur;

[0069] (B) having a base sequence capable of annealing to a regioncontaining a specific base in the target nucleic acid; and

[0070] (C) containing a sequence in which if there is a mismatch (or ifthere is no mismatch) between the specific base and a base correspondingto the specific base (i.e., that forms a hydrogen bond between thespecific base) in the Nucleotide in a complex composed of the Nucleotideand the target nucleic acid, the Nucleotide is not cleaved with anuclease, and if there is no mismatch (or if there is a mismatch)between the specific base and a base corresponding to the specific basein the Nucleotide, the Nucleotide is cleaved with a nuclease to generatea new 3′-terminus.

[0071] A fragment of a 5′ portion of the Nucleotide cleaved with anuclease can remain annealed to a target nucleic acid. Since a hydroxylgroup exists at the 3-position of ribose or deoxyribose at the3′-terminus of the fragment of the 5′ portion of the Nucleotide, a DNAcan be extended from the terminus by a DNA polymerase. Thus, theNucleotide serves as a precursor of a primer if it has a base sequencethat is cleavable with a nuclease.

[0072] As described above, the Nucleotide of the present invention ismodified at the 3′-terminus such that it cannot be utilized for a DNAextension reaction by a DNA polymerase. There is no specific limitationconcerning the means of modification as long as the above-mentionedobjects can be achieved. Examples thereof include addition, at the3′-terminus, of a dideoxy nucleotide, a nucleotide modified at thehydroxyl group at the 3-position of ribose, or a nucleotide withmodification that interferes with extension by a DNA polymerase due tosteric hindrance. Alkylation or other known modification methods can beutilized as a method for modifying the hydroxyl group, at the 3-positionof ribose of a nucleotide. For example, a DNA extension reaction can beprevented by aminoalkylation.

[0073] The Nucleotide of the present invention has a base sequencecapable of annealing, under conditions used, to a region in a targetnucleic acid for which a base substitution is to be detected. TheNucleotide has a sequence that is substantially complementary to atarget nucleic acid, and need not have a base sequence completelycomplementary to the target nucleic acid as long as the detection of asubstitution at the base of interest is not disturbed.

[0074] When the Nucleotide of the present invention is annealed to atarget nucleic acid and incubated in the presence of an appropriatenuclease and an appropriate DNA polymerase, cleavage of the Nucleotideis influenced by the presence of a base substitution in a target nucleicacid, that is, the presence of a mismatched site in a double-strandednucleic acid formed by annealing of the Nucleotide to a target nucleicacid. DNA extension using the target nucleic acid as a template occursonly if the Nucleotide is cleaved to generate a new 3′-terminus.Therefore, one can have information about the presence of a mismatch, orthe presence of a base substitution based on the presence of DNAextension.

[0075] According to the present invention, it is possible to prepare theNucleotide such that a mismatch is generated if there is a basesubstitution to be detected, and it is also possible to prepare theNucleotide such that a mismatch is not generated if there is a basesubstitution. Furthermore, one can have information about the presenceof a base substitution and the type of the substituted base at the sametime as follows: four types of Nucleotides each having one of four typesof bases placed at a position corresponding to the base of interest areprepared; and the type of the base contained in the primer that resultsin extension is then examined.

[0076] As described above, the Nucleotide of the present invention isconverted into a primer that is capable of DNA extension as a result ofcleavage with a nuclease. The portion of the Nucleotide 5′ to thecleavage site for the nuclease serves as a primer for DNA extension.There is no specific limitation concerning the nuclease as long as itcleaves (or does not cleave) the Nucleotide depending on the presence ofa mismatch in a double-stranded nucleic acid formed as a result ofannealing of the Nucleotide to a target nucleic acid. Examples thereofinclude a ribonuclease H, a restriction enzyme and a mismatch-specificnuclease.

[0077] A ribonuclease H (RNase H) is an enzyme that recognizes adouble-stranded nucleic acid composed of a DNA and an RNA andselectively cleaves the RNA strand. A Nucleotide that is cleaved with aribonuclease H only if there is no mismatch can be prepared by placing aribonucleotide at a site in the Nucleotide corresponding to the base forwhich a substitution is to be detected.

[0078] There is no specific limitation concerning the ribonuclease to beused according to the present invention as long as it has an activity ofrecognizing a double-stranded nucleic acid composed of the Nucleotide ofthe present invention containing a ribonucleotide and a DNAcomplementary thereto and selectively cleaving at the ribonucleotideportion. For example, a ribonuclease H from Escherichia coli, or aribonuclease H from a thermophilic bacterium belonging to genusBacillus, a bacterium belonging to genus Thermus, a bacterium belongingto genus Pyrococcus, a bacterium belonging to genus Thermotoga or abacterium belonging to genus Archaeoglobus can be preferably used assuch an enzyme. Although it is not intended to limit the presentinvention, the ribonuclease H preferably exhibits a high activity underthe same reaction conditions as those for a DNA polymerase to be used atthe same time. If the Nucleotide of the present invention is to be usedin combination with a nucleic acid amplification reaction, it ispreferable to use a ribonuclease H that exhibits its activity underconditions under which the reaction is carried out. For example, it isadvantageous to use a heat-resistant ribonuclease H if a nucleic acidamplification reaction that involves a reaction or treatment at a hightemperature (e.g., PCR) is to be utilized. For example, a ribonuclease Hfrom Bacillus caldotenax, Pyrococcus furiosus, Pyrococcus horikoshii,Thermococcus litoralis, Thermotoga maritima, Archaeoglobus fulgidus orMethanococcus jannashi can be used as a heat-resistant ribonuclease H.

[0079] A restriction enzyme is an enzyme that recognizes a specific basesequence (of 4 to 8 bases) in a DNA and cleaves at a position within oraround the sequence. If the base portion for which a substitution is tobe detected overlaps with a recognition sequence for a restrictionenzyme, a Nucleotide prepared to include the sequence can be used fordetection of a base substitution. If a mismatch is generated between aNucleotide and a target nucleic acid, cleavage with a restriction enzymedoes not occur. One can have information about the presence of the basesubstitution based on the results. If such a Nucleotide is to be used,it is necessary to make the target nucleic acid insusceptible tocleavage with the restriction enzyme. It is possible to conferresistance to the restriction enzyme specifically to the target nucleicacid, for example, by methylating the specific bases using amodification methylase corresponding to the restriction enzyme to beused.

[0080] An enzyme that recognizes and cleaves a mismatch between a targetnucleic acid and a Nucleotide unlike the above-mentioned two types ofnucleases may be used. Mut H or the like may be used as such an enzyme.

[0081] The Nucleotide of the present invention is cleaved with thenuclease, a new 3′-terminus is generated, and DNA extension is theninitiated from the terminus. There is no specific limitation concerningthe DNA polymerase used in this step as long as it is capable of DNAextension from the 3′-terminus of a primer depending on the sequence ofthe DNA as a template. Examples thereof include Escherichia coli DNApolymerase I, Klenow fragment, T7 DNA polymerase, DNA polymerases fromthermophilic bacteria belonging to genus Bacillus (Bst DNA polymerase,Bca DNA polymerase), DNA polymerases from bacteria belonging to genusThermus (Taq DNA polymerase, etc.) and α-type DNA polymerases fromthermophilic archaebacteria (Pfu DNA polymerase, etc.).

[0082] If the Nucleotide of the present invention is to be used incombination with a gene amplification reaction, a DNA polymerasesuitable for the gene amplification reaction is selected for use.

[0083] A fragment of a 3′ portion of the Nucleotide of the presentinvention generated as a result of cleavage with a nuclease can remainannealed to a target nucleic acid if it is sufficiently long, althoughit may be released from the target nucleic acid if it is short. If a DNApolymerase having a strand displacement activity is used, the fragmentis dessociated from the target nucleic acid upon DNA extension by theDNA polymerase. If a DNA polymerase having a 5′→3′ exonuclease activityis used, the fragment is degraded by the DNA polymerase.

[0084] Although it is not intended to limit the present invention, forexample, an oligonucleotide having a structure represented by thefollowing general formula can be used as the Nucleotide of the presentinvention in case where a ribonuclease H is used as a nuclease:

5′-dNa-Nb-dNc-N′-3′  General formula:

[0085] (a: an integer of 11 or more; b: an integer of 1 or more; c: 0 oran integer of 1 or more, dN: deoxyribonucleotide; N: ribonucleotide; N′:a nucleotide modified such that extension by a DNA polymerase does notoccur).

[0086] The portion represented by Nb in the general formula contains abase corresponding to the base as the subject of substitution detection.Furthermore, the Nucleotide may contain a nucleotide analog or aderivative (a modified nucleotide) as long as the function of theNucleotide is not spoiled.

[0087] A Nucleotide that is a chimeric oligonucleotide represented bythe general formula wherein N′ is a modified deoxyribonucleotide, a isan integer of 11 or more, b=1 to 3, c=0 to 2 is exemplified. There is nospecific limitation concerning the base corresponding to the base as thesubject of the base substitution detection as long as it is located inthe portion represented by Nb. In one embodiment of the presentinvention, for example, a Nucleotide in which the length of the portionrepresented by (dNc-N′) is three bases and a base corresponding to thebase for which a base substitution is to be detected is located at the3′ end of the portion represented by Nb can be preferably used. Such aNucleotide exhibits a good specificity in regard to detection of a basesubstitution.

[0088] Detection of a fragment of a 3′ portion released from theNucleotide of the present invention by cleavage with a nuclease or by aproduct generated upon a DNA extension reaction subsequent to thecleavage (an extension product) can be facilitated and the presence of abase substitution can be conveniently confirmed by appropriatelylabeling the Nucleotide.

[0089] There is no specific limitation concerning the method forlabeling a Nucleotide. For example, radioisotopes (³²P, etc.), dyes,fluorescent substances, luminescent substances, various ligands (biotin,digoxigenin, etc.) and enzymes can be used. The presence of a productderived from a labeled Nucleotide can be confirmed by a detection methodsuitable for the label. A ligand that cannot be directly detected may beused in combination with a ligand-binding substance having a detectablelabel. For example, a target nucleic acid can be detected with highsensitivity by using a product from a ligand-labeled Nucleotide incombination with an enzyme-labeled anti-ligand antibody and amplifyingthe signal.

[0090] Examples of embodiments of fluorescence labeled Nucleotidesinclude a Nucleotide labeled with both a fluorescent substance and asubstance having an action of quenching fluorescence emitted from thefluorescent substance with appropriate spacing. Such a primer does notemit fluorescence if it is in an intact state. However, it emitsfluorescence if it is cleaved with a nuclease, and the fluorescentsubstance and the quenching substance are placed at a distance. Sincesuch a Nucleotide emits fluorescence at the same time as the initiationof a DNA extension reaction, one can have information about the presenceof a base substitution by directly observing a reaction mixture during areaction.

[0091] (2) The Method for Detecting a Base Substitution of the PresentInvention

[0092] The Nucleotide of the present invention as described in (1) aboveis used in the method for detecting a base substitution of the presentinvention and the method comprises:

[0093] (1) mixing a sample containing a target nucleic acid with theNucleotide;

[0094] (2) treating the mixture with a nuclease and a DNA polymerase;and

[0095] (3) detecting the presence of cleavage of the Nucleotide with thenuclease. The presence of a base substitution is determined based on thepresence of cleavage of a Nucleotide with a nuclease according to thecharacteristics of the Nucleotide of the present invention as describedin (1) above.

[0096] A single-stranded or double-stranded nucleic acid (DNA or RNA)can be used as a target nucleic acid in the method for detecting a basesubstitution of the present invention. Depending on the nuclease to beused, it may be difficult to use an RNA as a target nucleic acid. Inthis case, a base substitution in an RNA can be detected by preparing acDNA using the RNA as a template and using the cDNA as a target nucleicacid.

[0097] According to the present invention, a sample containing a targetnucleic acid can be used for a detection reaction.

[0098] Any sample that may possibly contain a nucleic acid or anorganism such as a cell, a tissue (a biopsy sample, etc.), a wholeblood, a serum, a cerebrospinal fluid, a seminal fluid, a saliva, asputum, a urine, feces, a hair and a cell culture may be used withoutlimitation. Although it is not intended to limit the present invention,the test sample may be subjected to the method of the present inventionpreferably after it is appropriately processed, for example, after it isconverted into a form with which one can carry out a reaction using aDNA polymerase. Such processes include lysis of a cell as well asextraction and purification of a nucleic acid from a sample.

[0099] According to the method for detecting a base substitution of thepresent invention, the presence of a base substitution is determinedbased on the presence of cleavage of a Nucleotide to be used and thepresence of a DNA extension reaction subsequent to the cleavage. Thereis no specific limitation concerning the method for the determination,and known means of analyzing a nucleic acid can be used. Examples ofmethods for determining the presence of a DNA extension reaction includethe following: a method in which a generated extension product isseparated for confirmation by gel electrophoresis (agarose gel,polyacrylamide gel, etc.) or capillary electrophoresis; and a method inwhich increase in length of an extension product is measured by massspectrometry. In another embodiment, a method in which incorporation ofa nucleotide into an extension product is determined is exemplified. Inthis method, one can have information about an amount of a synthesizedextension product as an amount of a nucleotide triphosphate having anappropriate label incorporated into a macromolecular extension product.The amount of the generated extension product can be determined, forexample, after separating the product from unreacted nucleotides by acidprecipitation or gel electrophoresis. Furthermore, a method in whichpyrophosphate generated upon a DNA extension reaction is detected byenzymatic means may be used.

[0100] According to the detection method of the present invention, theextension product may be further amplified using a known nucleic acidamplification reaction. Such an embodiment is useful in view of highlysensitive detection of a base substitution.

[0101] Various nucleic acid amplification methods in which a primerhaving a sequence complementary to a nucleic acid as a template is usedcan be used as the nucleic acid amplification reaction withoutlimitation. For example, known amplification methods such as PolymeraseChain Reaction (PCR, U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159),Strand Displacement Amplification (SDA, JP-B 7-114718), Self-SustainedSequence Replication (3SR), Nucleic Acid Sequence Based Amplification(NASBA, Japanese Patent No. 2650159), Transcription-MediatedAmplification (TMA), and Isothermal and Chimeric primer-initiatedAmplification of Nucleic acids (ICAN, WO 00/56877) can be used. A basesubstitution in a target nucleic acid can be detected by using theNucleotide of the present invention as a primer for synthesis of a DNAcomplementary to a DNA strand as a template in such a method.

[0102] If the method for detecting a base substitution of the presentinvention is carried out utilizing the above-mentioned nucleic acidamplification method, the Nucleotide of the present invention is used asat least one of the primers used in the method, and a nuclease suitablefor the Nucleotide is included in the reaction system.

[0103] According to the detection of a base substitution utilizing anucleic acid amplification reaction as described above, the presence ofa base substitution can be determined based on generation of a specificamplification product by the reaction. Although it is not intended tolimit the present invention, for example, gel electrophoresis,hybridization using a probe having a sequence complementary to theamplification product, a fluorescence polarization method utilizing afluorescence labeled Nucleotide, the TaqMan method and the like can beused for the generated amplification product. In addition, detectionreactions suitable for the respective gene amplification methods can bealso utilized.

[0104] If base substitutions are to be analyzed using the detectionmethod of the present invention at a genomic level, the volume of thereaction system may be made smaller and a means of increasing degree ofintegration may be used in combination in order to analyze a largenumber of base sequences. A microchip sized several by severalcentimeters square to fingertip on which the basic processes of thedetection method or the analysis method of the present invention (e.g.,extraction of a DNA from a cell, a nucleic acid amplification reaction,detection of the DNA of interest, etc.) are integrated using anup-to-date microfabrication technique may be used in combination as sucha means. Optionally, processes of gel or capillary electrophoresis andhybridization with a detection probe may be combined. Such a system iscalled a microchip, a micro-capillary electrophoresis (CE) chip or ananochip.

[0105] Any nucleic acid amplification reaction may be utilized in such asystem as long as the DNA fragment of interest is amplified using thereaction. Although it is not intended to limit the present invention,for example, a method in which a nucleic acid can be amplified underisothermal conditions such as the ICAN method can be preferably used.The combination with such a method can simplify the system and is verypreferably utilized for the above-mentioned integrated system.Furthermore, a more highly integrated system can be constructedutilizing the techniques according to the present invention.

[0106] The specificity of detection of a base substitution can beimproved by including a modified nucleotide in the Nucleotide of thepresent invention and/or by appropriately adjusting the reactiontemperature in the method of the present invention.

[0107] The Nucleotide of the present invention having a label asdescribed in (1) above can facilitates confirmation of the presence of aDNA extension reaction, and is useful for the method for detecting abase substitution of the present invention. In this case, the presenceof an extension reaction is confirmed by detecting a labeled substancederived from the Nucleotide by a method suitable for the label asdescribed above.

[0108] For example, if the Nucleotide of the present invention to whicha fluorescent substance is attached is to be used and if the label isattached to a portion that is utilized as a primer, an extension productcan be detected utilizing the fluorescence. If a label is attached to aportion 3′ to the cleavage site for a nuclease in a Nucleotide, thepresence of an extension reaction can be detected based on dissociationof a 3′ fragment from the target nucleic acid, conversion of thefragment into a smaller molecule due to a 5′→3′ exonuclease activity ofa DNA polymerase or the like. A fluorescence polarization method ispreferably utilized for such an embodiment that involves change inmolecular weight of a fluorescence labeled Nucleotide.

[0109] If the Nucleotide of the present invention which is labeled byattaching a fluorescent substance and a substance having an action ofquenching fluorescence emitted from the fluorescent substance such thatthe fluorescence is not emitted is to be used, the fluorescence isemitted at the same time as the initiation of an extension reaction.Therefore, a base substitution can be very readily detected.

[0110] In the above-mentioned respective embodiments, by utilizingNucleotides each having adenine (A), cytosine (C), guanine (G), thymine(T) or uracil (U) at a position corresponding to the site for which abase substitution is to be detected as well as a distinguishabledifferent label, one can have information about the presence of a basesubstitution and the type of the substituted base at the same time.

[0111] The Nucleotide of the present invention can be used in a PCR fordetecting a base substitution. In this case, the Nucleotide of thepresent invention is used in place of one of PCR primers, and a nucleasesuitable for the Nucleotide is further added to a normal reactionmixture for PCR. A base substitution can be detected with highsensitivity by selecting a nuclease that is not inactivated under theconditions for the PCR.

[0112] Cells of higher animals including humans are usually diploidhaving a pair of chromosomes. Therefore, if a base substitution mayexist for a specific base on a chromosome, there are three possiblecases as follows: homozygote (homo-type) in which both chromosomes ofthe cell do not have a base substitution; homozygote (homo-type) inwhich base substitutions are present on both chromosomes; andheterozygote (hetero-type) in which only one of chromosomes has a basesubstitution.

[0113] It is possible to examine whether the genotype of a diploid cellor an individual having the cell is homo-type or hetero-type for aspecific base in a gene by applying the method for detecting a basesubstitution of the present invention to a nucleic acid sample preparedfrom the cell. Although it is not intended to limit the presentinvention, for example, if the method of the present invention iscarried out using Nucleotides that correspond to four types of bases andare cleaved if there is no mismatch, signals are detected as a result ofcleavage of the Nucleotides for two of the Nucleotides for a nucleicacid sample derived from a cell of which the genotype is hetero-type. Onthe other hand, a signal is detected for only one of the Nucleotides fora nucleic acid sample derived from a cell of which the genotype ishomo-type. In addition, it is possible to simultaneously determinewhether the homo-type has or does not have a base substitution. Asdescribed above, the method of the present invention is useful fordetection of a base substitution in an allele.

[0114] (3) The kit Used for Detecting a Base Substitution of the PresentInvention

[0115] The present invention provides a kit used for detection of a basesubstitution according to the present invention as described above. Inone embodiment, the kit contains the Nucleotide of the presentinvention. It may contain a set of Nucleotides each containing one offour types of bases that can be used to determine the presence of a basesubstitution and the type of the substituted base at the same time.Furthermore, the kit may contain a nuclease suitable for the Nucleotide,a DNA polymerase, a substrate for the DNA polymerase (dNTP), a buffersuitable for the reaction and the like. Alternatively, the kit maycontain a reagent for detection of a primer-extension product. A kitcontaining a reagent for preparing a reaction mixture used for a nucleicacid amplification method is preferable as a kit for detecting a basesubstitution in combination with a nucleic acid amplification method.

EXAMPLES

[0116] The following examples further illustrate the present inventionin detail but are not to be construed to limit the scope thereof.

Referential Example 1 Cloning of Pyrococcus furiosus RNase HII gene

[0117] (1) Preparation of Genomic DNA from Pyrococcus Furiosus

[0118] 2 L of a medium containing 1% Tryptone (Difco Laboratories), 0.5%yeast extract (Difco Laboratories), 1% soluble starch (Nacalai Tesque),3.5% Jamarine S Solid (Jamarine Laboratory), 0.5% Jamarine S Liquid(Jamarine Laboratory), 0.003% MgSO₄, 0.001% NaCl, 0.0001% FeSO₄·7H2O,0.0001% CoSO₄, 0.0001% CaCl₂·7H₂O, 0.0001% ZnSO₄, 0.1 ppm CuSO₄·5H₂O,0.1 ppm KAl (SO₄)₂, 0.1 ppm H₃BO₄, 0.1 ppm Na₂MoO₄·2H₂O and 0.25 ppmNiCl₂·6H₂O was placed in a 2-L medium bottle, sterilized at 120° C. for20 minutes, and bubbled with nitrogen gas to remove dissolved oxygen.Then, Pyrococcus furiosus (purchased from Deutsche Sammlung vonMikroorganismen; DSM3638) was inoculated into the medium and cultured at95° C. for 16 hours without shaking. After cultivation, cells werecollected by centrifugation.

[0119] The resulting cells were then suspended in 4 ml of 25% sucrose,50 mM Tris-HCl (pH 8.0). 0.4 ml of a 10 mg/ml lysozyme chloride (NacalaiTesque) aqueous solution was added thereto. The mixture was reacted at20° C. for 1 hour. After reaction, 24 ml of a mixture containing 150 mMNaCl, 1 mM EDTA and 20 mM Tris-HCl (pH 8.0), 0.2 ml of 20 mg/mlproteinase K (Takara Shuzo), and 2 ml of a 10% sodium lauryl sulfateaqueous solution were added to the reaction mixture. The mixture wasincubated at 37° C. for 1 hour.

[0120] After reaction, the mixture was subjected to phenol-chloroformextraction followed by ethanol precipitation to prepare about 1 mg ofgenomic DNA.

[0121] (2) Cloning of RNase HII Gene

[0122] The entire genomic sequence of Pyrococcus horikoshii waspublished [DNA Research, 5:55-76 (1998)]. The existence of a geneencoding a homologue of RNase HII (PH1650) in the genome was known (SEQID NO:1, the home page of National Institute of Technology andEvaluation: http://www/nite.go.jp/).

[0123] Homology between the PH1650 gene (SEQ ID NO:1) and the partiallypublished genomic sequence of Pyrococcus furiosus (the home page ofUniversity of Utah, Utah Genome Center:http://www.genome.utah.edu/sequence.html) was searched. As a result, ahighly homologous sequence was found.

[0124] Primers 1650Nde (SEQ ID NO:2) and 1650Bam (SEQ ID NO:3) weresynthesized on the basis of the homologous sequence.

[0125] A PCR was carried out in a volume of 100 μl using 200 ng of thePyrococcus furiosus genomic DNA obtained in Referential Example 1-(1) asa template, and 20 pmol of 1650Nde and 20 pmol of 1650Bam as primers.TaKaRa Ex Taq (Takara Shuzo) was used as a DNA polymerase for the PCRaccording to the attached protocol. The PCR was carried out as follows:30 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for1 minute. An amplified DNA fragment of about 0.7 kb was digested withNdeI and BamHI (both from Takara Shuzo). The resulting DNA fragment wasinserted between the NdeI site and the BamHI site in a plasmid vectorpET3a (Novagen) to make a plasmid pPFU220.

[0126] (3) Determination of Base Sequence of DNA Fragment ContainingRNase HII Gene

[0127] The base sequence of the DNA fragment inserted into pPFU220obtained in Referential Example 1-(2) was determined according to adideoxy method.

[0128] Analysis of the determined base sequence revealed an open readingframe presumably encoding RNase HII. The base sequence of the openreading frame is shown in SEQ ID NO:4. The amino acid sequence of RNaseHII deduced from the base sequence is shown in SEQ ID NO:5.

[0129]Escherichia coli JM109 transformed with the plasmid pPFU220 isdesignated and indicated as Escherichia coli JM109/pPFU220, anddeposited on Sep. 5, 2000 at International Patent Organism Depositary,National Institute of Advanced Industrial Science and Technology, AISTTsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken305-8566, Japan under accession number FERM P-18020 and at InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology under accession number FERM BP-7654 (date oftransmission to international depositary authority: Jul. 9, 2001).

[0130] (4) Preparation of Purified RNase HII Preparation

[0131]Escherichia coil HMS174(DE3) (Novagen) was transformed withpPFU220 obtained in Referential Example 1-(2). The resulting Escherichiacoli HMS174(DE3) harboring pPFU220 was inoculated into 2 L of LB mediumcontaining 100 μg/ml of ampicillin and cultured with shaking at 37° C.for 16 hours. After cultivation, cells collected by centrifugation weresuspended in 66.0 ml of a sonication buffer [50 mM Tris-HCl (pH 8.0), 1mM EDTA, 2 mM phenylmethanesulfonyl fluoride] and sonicated. Asupernatant obtained by centrifuging the sonicated suspension at 12000rpm for 10 minutes was heated at 60° C. for 15 minutes. It was thencentrifuged at 12000 rpm for 10 minutes again to collect a supernatant.Thus, 61.5 ml of a heated supernatant was obtained.

[0132] The heated supernatant was subjected to RESOURSE Q column(Amersham Pharmacia Biotech) equilibrated with Buffer A [50 mM Tris-HCl(pH 8.0), 1 mM EDTA] and chromatographed using FPLC system (AmershamPharmacia Biotech). As a result, RNase HII flowed through the RESOURSE Qcolumn.

[0133] 60.0 ml of the flow-through RNase HII fraction was subjected toRESOURSE S column (Amersham Pharmacia Biotech) equilibrated with BufferA and eluted with a linear gradient of 0 to 500 mM NaCl using FPLCsystem. A fraction containing RNase HII eluted with about 150 mM NaClwas obtained.

[0134] 2.0 ml of the RNase HII fraction was concentrated byultrafiltration using Centricon-10 (Amicon). 250 μl of the concentratewas subjected to Superdex 200 gel filtration column (Amersham PharmaciaBiotech) equilibrated with 50 mM Tris-HCl (pH 8.0) containing 100 mMNaCl and 0.1 mM EDTA and eluted with the same buffer. As a result, RNaseHII was eluted at a position corresponding to a molecular weight of 17kilodalton. This molecular weight corresponds to that of RNase HII in aform of a monomer.

[0135] The eluted RNase HII was used as a Pfu RNase HII preparation. AnRNase H activity was measured using the thus obtained Pfu RNase HIIpreparation as follows.

[0136] 10 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol (Nacalai Tesque),0.003% bovine serum albumin (fraction V, Sigma), 4% glycerol, 20 μg/mlpoly(dT) (Amersham Pharmacia Biotech) and 30 μg/ml poly(rA) (AmershamPharmacia Biotech) were mixed together. The mixture was incubated at 37°C. for 10 minutes and used as a substrate solution for measuring anRNase H activity. 1 μl of 1 M MnCl₂ was added to 100 μl of the substratesolution. The mixture was incubated at 40° C. An appropriate dilution ofthe Pfu RNase HII preparation was added to the mixture to initiate areaction. After reacting at 40° C. for 30 minutes, 10 μl of 0.5 M EDTAwas added thereto to terminate the reaction. Absorbance at 260 nm wasthen measured.

[0137] As a result, the value of absorbance at 260 nm for a reactionmixture in which the Pfu RNase HII preparation was added was higher thanthat for a reaction mixture in which 10 μl of 0.5 M EDTA was addedbefore the addition of the Pfu RNase HII preparation. Thus, it wasdemonstrated that the preparation had an RNase H activity.

[0138] (5) Measurement of Activity of Purified RNase H

[0139] (a) Preparation of Reagent Solutions Used

[0140] Reaction mixture for determining activity: The followingsubstances at the indicated final concentrations were contained insterile water: 40 mM Tris-HCl (pH 7.7 at 37° C.), 4 mM magnesiumchloride, 1 mM DTT, 0.003% BSA, 4% glycerol and 24 μM poly(dT).

[0141] Poly[8-³H]adenylic acid solution: 370 kBq of a poly[8-³H]adenylicacid solution was dissolved in 200 μl of sterile water.

[0142] Polyadenylic acid solution: Polyadenylic acid was diluted to aconcentration of 3 mM with sterile ultrapure water.

[0143] Enzyme dilution solution: The following substances at theindicated final concentrations were contained in sterile water: 25 mMTris-HCl (pH 7.5 at 37° C.), 5 mM 2-mercaptoethanol, 0.5 mM EDTA (pH 7.5at 37° C.), 30 mM sodium chloride and 50% glycerol.

[0144] Preparation of heat-denatured calf thymus DNA: 200 mg of calfthymus DNA was suspended and allowed to swell in 100 ml of TE buffer.The solution was diluted to a concentration of 1 mg/ml with sterileultrapure water based on the absorbance measured at UV 260 nm. Thediluted solution was heated at 100° C. for 10 minutes and then rapidlycooled in an ice bath.

[0145] (b) Method for Measuring Activity

[0146] 7 μl of the poly[8-³H]adenylic acid solution was added to 985 μlof the reaction mixture for determining activity prepared in (a) above.The mixture was incubated at 37° C. for 10 minutes. 8 μl of polyadenylicacid was added to the mixture to make the final concentration to 24 μM.The mixture was further incubated at 37° C. for 5 minutes. Thus, 1000 μlof a poly[8-³H]rA-poly-dT reaction mixture was prepared. 200 μl of thereaction mixture was then incubated at 30° C. for 5 minutes. 1 μl of anappropriate serial dilution of an enzyme solution was added thereto. 50μl each of samples was taken from the reaction mixture over time for usein subsequent measurement. The period of time in minutes from theaddition of the enzyme to the sampling is defined as Y. 50 μl of areaction mixture for total CPM or for blank was prepared by adding 1 μlof the enzyme dilution solution in place of an enzyme solution. 100 μlof 100 mM sodium pyrophosphate, 50 μl of the heat-denatured calf thymusDNA solution and 300 μl of 10% trichloroacetic acid (300 μl of ultrapurewater for measuring total CPM) were added to the sample. The mixture wasincubated at 0° C. for 5 minutes, and then centrifuged at 10000 rpm for10 minutes. After centrifugation, 250 μl of the resulting supernatantwas placed in a vial. 10 ml of Aquasol-2 (NEN Life Science Products) wasadded thereto. CPM was measured in a liquid scintillation counter.

[0147] (c) Calculation of Units

[0148] Unit value for each enzyme was calculated according to thefollowing equation.

Unit/ml={(measured CPM−blank CPM)×1.2*×20×1000×dilution rate}200(μl)/(total CPM×Y (min.)×50 (μl)×9**)

[0149] 1.2*: Amount in nmol of poly[8-³H]rA-poly-dT contained in totalCPM per 50 μl.

[0150] 9**: Correction coefficient.

Referential Example 2 Cloning of RNase HII Gene from Archaeoglobusfulgidus

[0151] (1) Preparation of Genomic DNA from Archaeoglobus Fulgidus

[0152] Cells of Archaeoglobus fulgidus (purchased from Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH; DSM4139) collected from 8 mlof a culture was suspended in 100 μl of 25% sucrose, 50 mM Tris-HCl (pH8.0). 20 μl of 0.5 M EDTA and 10 μl of a 10 mg/ml lysozyme chloride(Nacalai Tesque) aqueous solution was added thereto. The mixture wasreacted at 20° C. for 1 hour. After reaction, 800 μl of a mixturecontaining 150 mM NaCl, 1 mM EDTA and 20 mM Tris-HCl (pH 8.0), 10 μl of20 mg/ml proteinase K (Takara Shuzo) and 50 μl of a 10% sodium laurylsulfate aqueous solution were added to the reaction mixture. The mixturewas incubated at 37° C. for 1 hour. After reaction, the mixture wassubjected to phenol-chloroform extraction, ethanol precipitation andair-drying, and then dissolved in 50 μl of TE to obtain a genomic DNAsolution.

[0153] (2) Cloning of RNase HII Gene

[0154] The entire genomic sequence of the Archaeoglobus fulgidus hasbeen published [Klenk, H. P. et al., Nature, 390:364-370 (1997)]. Theexistence of one gene encoding a homologue of RNase HII (AF0621) wasknown (SEQ ID NO:13,http://www.tigr.org/tdb/CMR/btm/htmls/SplashPage.htlm).

[0155] Primers AfuNde (SEQ ID NO:14) and AfuBam (SEQ ID NO:15) weresynthesized on the basis of the sequence of the AF0621 gene (SEQ IDNO:13).

[0156] A PCR was carried out using 30 ng of the Archaeoglobus fulgidusgenomic DNA prepared in Referential Example 2-(1) as a template, and 20pmol of AfuNde and 20 pmol of AfuBam as primers in a volume of 100 μl.Pyrobest DNA polymerase (Takara Shuzo) was used as a DNA polymerase forthe PCR according to the attached protocol. The PCR was carried out asfollows: 40 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds and72° C. for 1 minute. An amplified DNA fragment of about 0.6 kb wasdigested with NdeI and BamHI (both from Takara Shuzo). The resulting DNAfragment was inserted between the NdeI site and the BamHI site in aplasmid vector pTV119Nd (a plasmid in which the NcoI site in pTV119N isconverted into a NdeI site) to make a plasmid pAFU204.

[0157] (3) Determination of Base Sequence of DNA Fragment ContainingRNase HII Gene

[0158] The base sequence of the DNA fragment inserted into pAFU204obtained in Referential Example 2-(2) was determined according to adideoxy method.

[0159] Analysis of the determined base sequence revealed an open readingframe presumably encoding RNase HII. The base sequence of the openreading frame is shown in SEQ ID NO:16. The amino acid sequence of RNaseHII deduced from the base sequence is shown in SEQ ID NO:17.

[0160]Escherichia coli JM109 transformed with the plasmid pAFU204 isdesignated and indicated as Escherichia coli JM109/pAFU204, anddeposited on Feb. 22, 2001 at International Patent Organism Depositary,National Institute of Advanced Industrial Science and Technology, AISTTsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken305-8566, Japan under accession number FERM P-18221 and at InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology under accession number FERM BP-7691 (date oftransmission to international depositary authority: Aug. 2, 2001).

[0161] (4) Preparation of Purified RNase HII Preparation

[0162]Escherichia coli JM109 was transformed with pAFU204 obtained inReferential Example 2-(2). The resulting Escherichia coli JM109harboring pAFU204 was inoculated into 2 L of LB medium containing 100μg/ml of ampicillin and cultured with shaking at 37° C. for 16 hours.After cultivation, cells collected by centrifugation were suspended in37.1 ml of a sonication buffer [50 mM-Tris-HCl (pH 8.0), 1 mM EDTA, 2 mMphenylmethanesulfonyl fluoride] and sonicated. A supernatant obtained bycentrifuging the sonicated suspension at 12000 rpm for 10 minutes washeated at 70° C. for 15 minutes. It was then centrifuged at 12000 rpmfor 10 minutes again to collect a supernatant. Thus, 40.3 ml of a heatedsupernatant was obtained.

[0163] The heated supernatant was subjected to RESOURSE Q column(Amersham Pharmacia Biotech) equilibrated with Buffer A [50 mM Tris-HCl(pH 8.0), 1 mM EDTA] and chromatographed using FPLC system (AmershamPharmacia Biotech). As a result, RNase HII flowed through the RESOURSE Qcolumn.

[0164] The flow-through RNase HII fraction was subjected to RESOURSE Scolumn (Amersham Pharmacia Biotech) equilibrated with Buffer A andchromatographed using FPLC system (Amersham Pharmacia Biotech). As aresult, RNase HII flowed through the RESOURSE S column.

[0165] 40.0 ml of the flow-through RNase HII fraction was subjected tothree rounds of dialysis against 2 L of Buffer B (50 mM Tris-HCl (pH7.0), 1 mM EDTA) containing 50 mM NaCl for 2 hours. 40.2 ml of thedialyzed enzyme solution was subjected to HiTrap-heparin column(Amersham Pharmacia Biotech) equilibrated with Buffer B containing 50 mMNaCl and eluted with a linear gradient of 50 to 550 mM NaCl using FPLCsystem. As a result, a fraction containing RNase HII eluted with about240 mM NaCl was obtained.

[0166] 7.8 ml of the RNase HII fraction was concentrated byultrafiltration using Centricon-10 (Amicon). Four portions separatedfrom about 600 μl of the concentrate were subjected to Superose 6 gelfiltration column (Amersham Pharmacia Biotech) equilibrated with 50 mMTris-HCl (pH 7.0) containing 100 mM NaCl and 0.1 mM EDTA and eluted withthe same buffer. As a result, RNase HII was eluted at a positioncorresponding to a molecular weight of 30.0 kilodalton. This molecularweight corresponds to that of RNase HII in a form of a monomer.

[0167] The RNase HII eluted as described above was used as an Afu RNaseHII preparation.

[0168] An enzymatic activity was measured as described in ReferentialExample 1-(5) using the thus obtained Afu RNase HII preparation. As aresult, an RNase H activity was observed for the Afu RNase HIIpreparation.

[0169] Unit value of a heat-resistant RNase H in the following Exampleswas calculated as follows.

[0170] 1 mg of poly(rA) or poly(dT) (both from Amersham PharmaciaBiotech) was dissolved in 1 ml of 40 mM Tris-HCl (pH 7.7) containing 1mM EDTA to prepare a poly(rA) solution and a poly(dT) solution.

[0171] The poly(rA) solution (to a final concentration of 20 μg/ml) andthe poly(dT) solution (to a final concentration of 30 μg/ml) were thenadded to 40 mM Tris-HCl (pH 7.7) containing 4 mM MgCl₂, 1 mM DTT, 0.003%BSA and 4% glycerol. The mixture was reacted at 37° C. for 10 minutesand then cooled to 4° C. to prepare a poly(rA)-poly(dT) solution. 1 μlof an appropriately diluted enzyme solution was added to 100 μl of thepoly(rA)-poly(dT) solution. The mixture was reacted at 40° C. for 10minutes. 10 μl of 0.5 M EDTA was added thereto to terminate thereaction. Absorbance at 260 nm was then measured. As a control, 10 μl of0.5 M EDTA was added to the reaction mixture, the resulting mixture wasreacted at 40° C. for 10 minutes, and the absorbance was then measured.A value (difference in absorbance) was obtained by subtracting theabsorbance for the control from the absorbance for the reaction in theabsence of EDTA. Thus, the concentration of nucleotide released frompoly(rA)-poly(dT) hybrid-by the enzymatic reaction was determined on thebasis of the difference in absorbance. One unit of an RNase H wasdefined as an amount of enzyme that increases A₂₆₀ corresponding torelease of 1 nmol of ribonucleotide in 10 minutes, which was calculatedaccording to the following equation:

Unit=[Difference in Absorbance×Reaction Volume(ml)]/0.0152×(110/100)×Dilution Rate

Example 1 Detection of Base Substitution in Human c-Ki-ras Gene

[0172] (1) Preparation of Template

[0173] DNA fragments each having a sequence GGT (Gly), CGT (Arg), TGT(Cys) or AGT (Ser) for codon 12 in human c-Ki-ras exon 1 were prepared.Briefly, amplification products obtained by PCRs using template DNAscorresponding to the above-mentioned codons in ras Mutant Set c-Ki-rascodon 12 (Takara Shuzo) and ras Gene Primer Set c-Ki-ras/12 (TakaraShuzo) were cloned into a vector pT7-Blue (Novagen). PCRs using the thusobtained recombinant plasmids as templates and M13 primers M4 and RV(both from Takara Shuzo) were carried out. The resulting amplifiedfragments were recovered and designated as templates 12G, 12R, 12C and12S, respectively.

[0174] (2) Detection of Base Substitution

[0175] Three chimeric oligonucleotides having base sequences of SEQ IDNOS:7 to 9 as forward Nucleotides for specifically detecting thetemplate 12G were synthesized on the basis of the base sequence of humanc-Ki-ras exon 1. The hydroxyl group at the 3-position of ribose moietyof the nucleotide at the 3′ end of each chimeric oligonucleotide wasmodified with aminohexyl. Each of the Nucleotides had a sequencecomplementary to the base sequence of human c-Ki-ras exon 1 in whichcodon 12 encoded Gly. An oligonucleotide having a base sequence of SEQID NO:6 was synthesized as an antisense primer for nucleic acidamplification.

[0176] A reaction mixture of a total volume of 5 μl containing 50 pmoleach of the forward Nucleotide and the antisense primer, 1 μl of a 0.25%propylenediamine aqueous solution and 1 μg of one of the templates 12G,12C, 12R and 12S as a template was prepared. The forward Nucleotide andthe antisense primer were annealed to the template by heating at 98° C.for 2 minutes and then at 53° C. in Thermal Cycler Personal (TakaraShuzo). 20 μl of a mixture containing 0.625 mM dNTP mix, 40 mM Hepes-KOHbuffer (pH 7.8), 125 mM potassium acetate, 5 mM magnesium acetate,0.0125% bovine serum albumin, 1.25% dimethyl sulfoxide, 16 U of PfuRNase HII as described in Referential Example 1, 5.5 U of BcaBest DNApolymerase (Takara Shuzo) and sterile water was added to the heatedmixture to make the final volume to 25 μl. The reaction mixture wasincubated at 53° C. for 1 hour. After reaction, 5 μl each of thereaction mixtures was subjected to electrophoresis on 3.0% agarose gel.The results are shown in FIG. 1. The reaction mixtures in which thetemplates 12G, 12C, 12R and 12S were used were applied to lanes 1 to 4in the agarose gels as shown in FIGS. 1-1, 1-2 and 1-3, respectively.FIGS. 1-1, 1-2 and 1-3 show results for the reactions in which theNucleotides of SEQ ID NOS:7, 8 and 9 were used; respectively.

[0177] As shown in FIG. 1, using the Nucleotides of SEQ ID NOS:7-9,amplification products were observed only when the template 12G wasused, that is, when the target nucleic acid encoded Gly for codon 12.These results show that a base substitution in a target nucleic acid canbe distinguished by using the Nucleotide of the present invention.Furthermore, it was confirmed that specific amplification could beimproved by using a Nucleotide containing inosine.

Example 2 Detection of other Alleles for c-Ki-ras Codon 12

[0178] Based on the results of Example 1, chimeric oligonucleotideshaving base sequences of SEQ ID NOS:10 to 12 were synthesized asNucleotides capable of specifically distinguishing the bases of codon 12in 12R, 12C and 12S prepared in Example 1-(1). SEQ ID NOS:10, 11 and 12show base sequences corresponding to alleles in which codon 12 encodesCys, Arg and Ser, respectively. The hydroxyl group at the 3-position ofribose moiety of the nucleotide at the 3′ end of each Nucleotide wasmodified with aminohexyl. Reactions were carried out using theseNucleotides and the antisense primer of SEQ ID NO:6 under reactionconditions as described in Example 1-(2). After reaction, 5 μl each ofthe reaction mixtures was subjected to electrophoresis on 3.0% agarosegel. The results are shown in FIG. 2. The reaction mixtures in which thetemplates 12G, 12C, 12R and 12S were used were applied to lanes 1 to 4in the agarose gels as shown in FIGS. 2-1, 2-2 and 2-3, respectively.FIGS. 2-1, 2-2 and 2-3 show results for the reactions in which theNucleotides of SEQ ID NOS:10, 11 and 12 were used, respectively.

[0179] As shown in FIG. 2, specific amplification products were observedonly when the Nucleotides of SEQ ID NOS:10, 11 and 12 were used incombination with the templates 12C, 12R and 12S, respectively. Thus, theNucleotides of the present invention could exactly distinguish the basesof interest. Furthermore, it was confirmed that specific amplificationcould be improved by using an oligonucleotide containing inosine.

Example 3 Allele-Specific DNA Amplification of Genomic DNA

[0180] Reactions were carried out under conditions as described in (2)above. In the reaction, 150 ng or 30 ng of a human genomic DNA(Clontech) for which it had been confirmed that codon 12 in c-Ki-rasexon 1 encodes Gly (GGT), the Nucleotides of SEQ ID NOS:7, 10, 11 and 12(corresponding to Gly, Cys, Arg and Ser at codon 12, respectively) whichhad been demonstrated to be able to specifically detect the four allelesfor codon 12 in Example 1 and 2, as well as the antisense primer of SEQID NO:6 were used. After reaction, 5 μl each of the reaction mixtureswas subjected to electrophoresis on 3.0% agarose gel. The results areshown in FIG. 3.

[0181] The reaction mixtures in which the Nucleotide of SEQ ID NOS:7,10, 11 and 12 were used were applied to lanes 1 to 4 in the agarose gelsas shown in FIGS. 3-1 and 3-2, respectively. FIGS. 3-1 and 3-2 showresults for the reactions in which 150 ng and 30 ng of the human genomicDNA were used, respectively.

[0182] As shown in FIG. 3, amplification of a DNA fragment was observedonly when the Nucleotide of SEQ ID NO:7 was used regardless of theamount of the human genomic DNA, whereas amplification of a DNA fragmentwas not observed using other Nucleotides. These results confirmed thatthe method for detecting a base substitution of the present inventioncould be used to detect a specific allele in a genomic DNA.

Example 4 Detection Using Various RNase H's

[0183] Use of various RNase H's in the detection of a base substitutionas described in Example 1 was examined.

[0184] Specifically, Afu RNase HII as described in Referential Example 2or Mja RNase HII, an RNase H derived from Methanococcus jannashi,prepared as described in Structure, 8:897-904 was used in place of PfuRNase HII. Reactions were carried out under conditions as described inExample 1 using the Nucleotide of SEQ ID NO:7 as a forward Nucleotideand the oligonucleotide of SEQ ID NO:6 as an antisense primer. Afterreaction, 5 μl each of the reaction mixtures was subjected toelectrophoresis on 3.0% agarose gel. The results are shown in FIG. 4.The reaction mixtures in which the templates 12G, 12C, 12R and 12S wereused were applied to lanes 1 to 4 in the agarose gels as shown in FIGS.4-1 and 4-2, respectively. FIGS. 4-1 and 4-2 show results for thereactions in which Afu RNase HII and Mja RNase HII were used,respectively.

[0185] As shown in FIG. 4, using Afu RNase HII and Mja RNase HII,amplification products were observed only when the template 12G wasused, that is, when the target nucleic acid encoded Gly for codon 12.These results show that a base substitution in a target nucleic acid canbe distinguished using these RNase H's.

Example 5 Detection of SNP using DNA Amplification Reaction System (PCR)that Requires Denaturation Step

[0186] The method of the present invention was examined using a DNAamplification reaction system that requires a denaturation step. Achimeric oligonucleotide of SEQ ID NO:25 was synthesized as a senseNucleotide for specifically detecting the template 12G on the basis ofthe base sequence of human c-Ki-ras exon 1. The hydroxyl group at the3-position of ribose moiety of the nucleotide at the 3′ end of theNucleotide was modified with aminohexyl. A primer having the basesequence of SEQ ID NO:18 was synthesized as an antisense primer as well.A reaction mixture of total volume of 24 μl containing 50 pmol each ofthe synthetic Nucleotide and the primer (the sense Nucleotide and theantisense primer), 2.5 μl of Ex Taq buffer (Takara Shuzo), 2 μl of 2.5mM dNTP mix, 50 U of Afu RNase HII and 0.625 U of Ex Taq DNA polymerase(Takara Shuzo) was prepared. 1 μl of a 10 ng/ul solution of the template12G, 12C, 12R or 12S prepared in Example 1 was added to the reactionmixture. A PCR was carried out using Thermal Cycler (Takara Shuzo) asfollows: 25 or 30 cycles of 94° C. for 5 seconds, 59° C. for 2 minutesand 72° C. for 5 seconds. After reaction, 1 μl each of the reactionmixtures was analyzed using Agilent 2100 Bioanalyzer (Hewlett-Packard).The results are shown in FIG. 5. FIG. 5 is a graph that illustrates theamounts of amplification products of interest for the respectivetemplates. The vertical axis represents the amount of amplificationproduct of interest and the horizontal axis represents the PCR cyclenumber. As shown in FIG. 5, specific amplification of the DNA ofinterest was observed only when the template 12G, of which the allelewas consistent with the primer used for detection, was used. Thus, itwas confirmed that the method of the present invention was alsoeffective for a DNA amplification reaction system that requires a stepof denaturing a nucleic acid as a template.

Example 6 Allele-Specific Detection of K-ras Codon 61

[0187] Detection of another base substitution was examined.Specifically, DNA fragments each having a sequence CAA (Glu), AAA (Lys)or GAA (Gln) for codon 61 in human c-Ki-ras exon 2 amplified by PCRsusing the DNA primers of SEQ ID NOS:19 and 20 were cloned into thevector pT7-Blue. The vectors into which the DNA fragments were clonedwere purified according to a conventional method and designated as 61Q,61K and 61E, respectively. Based on the results of Example 1-(2),chimeric oligonucleotides of SEQ ID NOS:21, 22 and 23 were synthesizedas Nucleotides for specifically detecting the respective vectors 61Q,61K and 61E on the basis of the base sequence of human c-Ki-ras exon 2.The hydroxyl group at the 3-position of ribose moiety of the nucleotideat the 3′ end of each Nucleotide was modified with aminohexyl. Thefollowing reaction was carried out using the Nucleotide as a senseprimer and the primer of SEQ ID NO:24 as an antisense primer. A reactionmixture of a total volume of 5 μl containing 50 pmol each of thesynthetic oligonucleotide primers (sense and antisense primers), 1 μl ofa 0.05% propylenediamine aqueous solution and 10 pg of one of thetemplate DNAs 61Q, 61K and 61E was prepared. The primers were annealedto the template by heating at 98° C. for 2 minutes and then at 53° C. inThermal Cycler Personal (Takara Shuzo). 20 μl of a mixture containing0.625 mM dNTP mix; 40 mM Hepes-KOH buffer (pH 7.8), 125 mM potassiumacetate, 5 mM magnesium acetate, 0.0125% bovine serum albumin, 1.25%dimethyl sulfoxide, 11 U of Afu RNase HII (Takara Shuzo), 5.5 U ofBcaBest DNA polymerase (Takara Shuzo) and sterile water was added to theheated mixture to make the final volume to 25 μl. The reaction mixturewas incubated at 58° C. for 1 hour. After reaction, 5 μl each of thereaction mixtures was subjected to electrophoresis on 3.0% agarose gel.The results are shown in FIG. 6. FIG. 6A is an electrophoresis patternthat represents results for detection using the primer of SEQ ID NO:21for detecting 61Q. Lanes 1, 2 and 3 represent results obtained using thetemplates 61Q, 61K and 61E as templates, respectively. FIG. 6B is anelectrophoresis pattern that represents results for detection using theprimer of SEQ ID NO:22 for detecting 61K. Lanes 1, 2 and 3 representresults obtained using the templates 61Q, 61K and 61E, respectively.FIG. 6C is an electrophoresis pattern that represents results fordetection obtained using the primer of SEQ ID NO:23 for detecting 61E.Lanes 1, 2 and 3 represent results obtained using the templates 61Q, 61Kand 61E, respectively.

[0188] As shown in FIGS. 6A, 6B and 6C, it was confirmed that the DNAamplification products of interest were obtained by ICAN reactions in anallele-specific manner using SEQ ID NOS:21, 22 and 23. Thus, it wasconfirmed that the method of the present invention was effective if theobjective base substitution was changed.

Example 7 Allele-Specific Detection of CYP2C19(636)

[0189] (1) A detection method for distinguishing genetic homo-type fromhetero-type was examined. The allele for the 636th base in human CYP2C19was selected as a subject. First, DNA fragments in which the 636 th basein human CYP2C19 was G or A amplified by PCRs using DNA primers of SEQID NOS:26 and 27 were cloned into the vector pT7-Blue. The plasmids intowhich these DNA fragments were cloned were purified according to aconventional method and designated as plasmids 636G and 636A.

[0190] The plasmids 636G and 636A as well as a plasmid 636G/A preparedby mixing the plasmids 636G and 636A at 1:1 were used as templates. Theplasmids 636G and 636A served as models for genetic homo-type, whereasthe plasmid 636G/A served as a mode& for genetic hetero-type. Next,Nucleotide of SEQ ID NOS:28 and 29 were synthesized as Nucleotides forspecifically detecting the 636G and the 636A, respectively. Thefollowing reaction was carried out using the Nucleotide as a senseprimer and the primer of SEQ ID NO:30 as an antisense primer. A reactionmixture of a total volume of 5 μl containing 50 pmol each of thesynthetic oligonucleotide primers (sense and antisense primers), 1 μl ofa 0.05% propylenediamine aqueous solution and 1 pg of one of theplasmids 636G, 636A and 636G/A as a template DNA was prepared. Theprimers were annealed to the template by heating at 98° C. for 2 minutesand then at 53° C. in Thermal Cycler Personal (Takara Shuzo). 20 μl of amixture containing 0.625 mM dNTP mix, 40 mM Hepes-KOH buffer (pH 7.8),125 mM potassium acetate, 5 mM magnesium acetate, 0.0125% bovine serumalbumin, 1.25% dimethyl sulfoxide, 11 U of Afu RNase HII, 5.5 U ofBcaBest DNA polymerase and sterile water was added to the heated mixtureto make the final volume to 25 μl. The reaction mixture was incubated at53° C. for 1 hour. After reaction, 5 μl each of the reaction mixtureswas subjected to electrophoresis on 3.0% agarose gel. The results areshown in FIGS. 7A and 7B. FIG. 7A is an electrophoresis pattern thatrepresents results for detection using the Nucleotide 636G. Lanes 1, 2and 3 represent results obtained using the plasmids 636G, 636A and636G/A as templates, respectively.

[0191]FIG. 7B is an electrophoresis pattern that represents results fordetection using the Nucleotide 636A. Lanes 1, 2 and 3 represent resultsobtained using the plasmids 636G, 636A and 636G/A as templates,respectively. As shown in FIGS. 7A and 7B, it was confirmed thatdetection could be carried out in an allele-specific manner using theNucleotides.

[0192] (2) A human genomic DNA was used as a template in comparison withPCR-RFLP for analysis. SNP typing was carried out as described in (1)above using 150 ng of a human genomic DNA (Clontech) as a template. Theresults are shown in FIG. 7C. FIG. 7C is a electrophoresis pattern thatrepresents results for SNP typing of the human genomic DNA. Lanes 1 and2 and represent results obtained using the Nucleotides 636G and 636A,respectively.

[0193] As shown in FIG. 7C, the amplified DNA of interest was detectedonly when the Nucleotide 636G was used. The allele for the 636th base inCYP2C19 of the genomic DNA was determined to be homo-type (636G/G).

[0194] On the other hand, typing was carried out by PCR-RFLP using thehuman genomic DNA. A PCR was carried out using 150 ng of the genomic DNAand primers of SEQ ID NOS:26 and 27. The resulting PCR amplificationproduct was treated with BamHI and the reaction mixture was subjected toelectrophoresis on 3.0% agarose gel. The results are shown in FIG. 7D.FIG. 7D is an electrophoresis pattern that represents results for typingby PCR-RFLP using the human genomic DNA as a template. Lanes 1 and 2represent results for the PCR amplification product and the PCRamplification product digested with BamHI, respectively.

[0195] As shown in FIG. 7D, the PCR amplification product was completelydigested with BamHI. Thus, the allele for the 636th base in CYP2Cl9 inthe genomic DNA was also determined to be homo-type (636G/G) byPCR-RFLP. It was confirmed that the results obtained using the methodfor detecting a base substitution of the present invention wereconsistent with those obtained using the conventional SNP typing byPCR-RFLP.

[0196] (3) Using the plasmids 636G, 636A, 636G/A prepared in (1) above,a detection method was examined assuming genotype of a homologouschromosome. The reaction was carried out as follows. First, theNucleotides 636G and 636A having fluorescence labels Rox (ABI) and Fam(ABI) being attached at the 5′-termini which are distinguishable eachother were synthesized. A mixture containing equal amounts of thefluorescence labeled Nucleotides was used. Detection was carried out asdescribed in (1) above. After reaction, a portion of each reactionmixture was subjected to electrophoresis on 3.0% agarose gel to fullyseparate the amplification product from the unreacted fluorescencelabeled Nucleotide. After electrophoresis, the agarose gel was analyzedusing FM-BIO II Multi-View (Takara Shuzo). As a result, when the plasmid636G was used as a template, only the fluorescence signal from thefluorescent label Rox was observed. When the plasmid 636A was used as atemplate, only the fluorescence signal from the fluorescent label Famwas observed. Furthermore, when the plasmid 636G/A was used as atemplate, the fluorescence signals from both Rox and Fam were observed.Based on these results, it was confirmed that the method of the presentinvention was useful as a method that could be used to analyze thegenotype (homo-type or hetero-type) on a homologous chromosome.

Example 8 Typing Using Genomic DNA Extracted from Whole Blood

[0197] A genomic DNA was prepared using Dr. GenTLE™ (Takara Shuzo) from200 μl each of whole blood samples 1-6 collected from healthyindividuals after obtaining informed consent. SNP typing was carried outas described in Example 7-(1) using 160 ng of the prepared genomic DNAas a template as well as the Nucleotides of SEQ ID NOS:28 and 29 asprimers for specifically detecting the alleles 636G and 636A. Theresults are shown in FIGS. 8A-F. FIGS. 8A-F are electrophoresis patternsthat represent results for typings carried out as described in Example7-(1) using genomic DNAs extracted from the blood samples 1-6 astemplates. Lanes 1 and 2 represent results obtained using theNucleotides of SEQ ID NO:28 (for detecting 636G) and SEQ ID NO:29 (fordetecting 636A), respectively. Based on the patterns of amplificationproducts as shown in FIGS. 8A-F, the alleles of the respective bloodsamples for the 636th base in CYP2Cl9 were typed as follows (1: G/A, 2:G/G, 3: G/A, 4: G/G, 5: G/G, 6: G/G). On the other hand, typing byPCR-RFLP was carried out as described in Example 7-(2) using the samegenomic DNA as a template. The results are shown in FIG. 8G. FIG. 8G isan electrophoresis pattern that represents results for typing byPCR-RFLP using genomic DNAs prepared from the blood samples 1-6 astemplates. Lanes 1-6 represent results obtained using the genomic DNAsextracted from the blood samples 1-6 as templates, respectively. Basedon the results of electrophoresis as shown in FIG. 8G which shows thecleavage pattern of the PCR amplification products obtained using theDNAs prepared from the respective blood samples as templates, thealleles for the 636th base in CYP2C19 were typed as follows (1: G/A, 2:G/G, 3: G/A, 4: G/G, 5: G/G, 6: G/G), which were consistent with thoseas described above.

[0198] As described above, it was confirmed that the method of thepresent invention was also effective when a practical clinical testsample was used.

[0199] Industrial Applicability

[0200] The Nucleotide of the present invention and the method fordetecting a base substitution using said Nucleotide as described aboveare useful for detecting a naturally occurring or artificiallyintroduced base substitution.

[0201] According to the present invention, the presence of a basesubstitution in a target nucleic acid can be detected conveniently withreproducibility. The method of the present invention can be readilycombined with a known nucleic acid amplification method, and can be usedto detect a base substitution with high sensitivity. Furthermore, byusing Nucleotides having appropriate sequences in combination, it ispossible to have information about the presence of a base substitutionand the type of the substituted base at the same time.

[0202] The present invention can be used for detecting or identifying abase substitution (e.g., SNP) generated in a genomic DNA of an organismsuch as a polymorphism or a variation. Thus, the present invention isuseful in fields of genomic drug development and genomic medicine forsearching for a disease gene in humans, analysis of drug resistance orthe like.

[0203] Sequence Listing Free Text

[0204] SEQ ID NO:1: a gene encoding a polypeptide having a RNaseHIIactivity from Pyrococcus horikoshii

[0205] SEQ ID NO:2: PCR primer 1650Nde for cloning a gene encoding apolypeptide having a RNase HII activity from Pyrococcus furiosus

[0206] SEQ ID NO:3: PCR primer 1650Bam for cloning a gene encoding apolypeptide having a RNaseHII activity from Pyrococcus furiosus

[0207] SEQ ID NO:6: Chimeric oligonucleotide primer to amplify the DNAof a portion of human c-Ki-ras gene. “nucleotides 18 to 20 areribonucleotides-other nucleotides are deoxyribonucleotides”

[0208] SEQ ID NO:7: Chimeric oligonucleotide to detect the nucleotidesubstitution on human c-Ki-ras gene. “nucleotides 13 to 15 areribonucleotides-other nucleotides are deoxyribonucleotides and the 3′-OHgroup of the nucleotide at 3′ end is protected with amino hexyl group”

[0209] SEQ ID NO:8: Chimeric oligonucleotide primer precursor to detectthe nucleotide substitution on human c-Ki-ras gene. “nucleotides 12 to15 are ribonucleotides, nucleotide 17 is inosine-other nucleotides aredeoxyribonucleotides and the 3′-OH-group of the nucleotide at 3′ end isprotected with amino hexyl group”

[0210] SEQ ID NO:9: Chimeric oligonucleotide to detect the nucleotidesubstitution on human c-Ki-ras gene. “nucleotides 14 and 15 areribonucleotides-other nucleotides are deoxyribonucleotides and the 3′-OHgroup of the nucleotide at 3′ end is protected with amino hexyl group”

[0211] SEQ ID NO:10: Chimeric oligonucleotide to detect the nucleotidesubstitution on human c-Ki-ras gene. “nucleotides 13 to 15 areribonucleotides-other nucleotides are deoxyribonucleotides and the 3′-OHgroup of the nucleotide at 3′ end is protected with amino hexyl group”

[0212] SEQ ID NO:11: Chimeric oligonucleotide to detect the nucleotidesubstitution on human c-Ki-ras gene. “nucleotides 13 to 15 areribonucleotides-other nucleotides are deoxyribonucleotides and the 3′-OHgroup of the nucleotide at 3′ end is protected with amino hexyl group”

[0213] SEQ ID NO:12: Chimeric oligonucleotide to detect the nucleotidesubstitution on human c-Ki-ras gene. “nucleotides 13 to 15 areribonucleotides, nucleotide 17 is inosine-other nucleotides aredeoxyribonucleotides and the 3′-OH group of the nucleotide at 3′ end isprotected with amino hexyl group”

[0214] SEQ ID NO:13: Base sequence of AF0621 gene from Archaeoglobusfulgidus.

[0215] SEQ ID NO:14: PCR primer AfuNde for cloning a gene encoding apolypeptide having a RNaseHII activity from Archaeoglobus fulgidus.

[0216] SEQ ID NO:15: PCR primer AfuBam for cloning a gene encoding apolypeptide having a RNaseHII activity from Archaeoglobus fulgidus.

[0217] SEQ ID NO:16: Base sequence of ORF in RnaseHII from Archaeoglobusfulgidus.

[0218] SEQ ID NO:17: Amino acid sequence of RNaseHII from Archaeoglobusfulgidus.

[0219] SEQ ID NO:18: Designed PCR primer to amplify a portion ofc-ki-ras oncogene exon 1

[0220] SEQ ID NO:19 Designed PCR primer to amplify a portion of humanc-ki-ras oncogene exon 2

[0221] SEQ ID NO:20: Designed PCR primer to amplify a portion of humanc-ki-ras oncogene exon 2

[0222] SEQ ID NO:21: Chimeric oligonucleotide to detect the nucleotidesubstitution on human c-Ki-ras gene. “nucleotides 13 to 15 areribonucleotides-other nucleotides are deoxyribonucleotides and the 3′-OHgroup of the nucleotide at 3′ end is protected with amino hexyl group”

[0223] SEQ ID NO:22: Chimeric oligonucleotide to detect the nucleotidesubstitution on human c-Ki-ras gene. “nucleotides 13 to 15 areribonucleotides-other nucleotides are deoxyribonucleotides and the 3′-OHgroup of the nucleotide at 3′ end is protected with amino hexyl group”

[0224] SEQ ID NO:23: Chimeric oligonucleotide to detect the nucleotidesubstitution on human c-Ki-ras gene. “nucleotides 13 to 15 areribonucleotides-other nucleotides are deoxyribonucleotides and the 3′-OHgroup of the nucleotide at 3′ end is protected with amino hexyl group”

[0225] SEQ ID NO:24: Chimeric oligonucleotide to detect the nucleotidesubstitution on human c-Ki-ras gene. “nucleotides 17 to 19 areribonucleotides-other nucleotides are deoxyribonucleotides”

[0226] SEQ ID NO:25: Chimeric oligonucleotide to detect the nucleotidesubstitution on human c-Ki-ras gene. “nucleotides 16 to 18 areribonucleotides-other nucleotides are deoxyribonucleotides and the 3′-OHgroup of the nucleotide at 3′ end is protected with amino hexyl group”

[0227] SEQ ID NO:26: Designed PCR primer to amplify a portion of humanCYP2C19 gene

[0228] SEQ ID NO:27: Designed PCR primer to amplify a portion of humanCYP2C19 gene

[0229] SEQ ID NO:28: Chimeric oligonucleotide to detect the nucleotidesubstitution on human CYP2C19 gene. “nucleotides 13 to 15 areribonucleotides-other nucleotides are deoxyribonucleotides and the 3′-OHgroup of the nucleotide at 3′ end is protected with amino hexyl group”

[0230] SEQ ID NO:29: Chimeric oligonucleotide to detect the nucleotidesubstitution on human CYP2C19 gene. “nucleotides 13 to 15 areribonucleotides-other nucleotides are deoxyribonucleotides and the 3′-OHgroup of the nucleotide at 3′ end is protected with amino hexyl group”

[0231] SEQ ID NO:30: Chimeric oligonucleotide primer to amplify aportion of human CYP2Cl9 gene. “nucleotides 19 to 21 areribonucleotides-other nucleotides are deoxyribonucleotides”

1. A method for detecting the presence of a base substitution at aspecific base in a target nucleic acid, the method comprising: (1)mixing a sample containing a target nucleic acid with a Nucleotide,wherein the Nucleotide (A) is modified at the 3′-terminus such thatextension from the terminus by a DNA polymerase does not occur; (B) hasa base sequence capable of annealing to a region containing a specificbase in the target nucleic acid; and (C) contains a sequence in which ifthere is a mismatch between the specific base and a base correspondingto the specific base in the Nucleotide in a complex composed of theNucleotide and the target nucleic acid, the Nucleotide is not cleavedwith a nuclease, and if there is no mismatch between the specific baseand a base corresponding to the specific base in the Nucleotide, theNucleotide is cleaved with a nuclease to generate a new 3′-terminus; (2)treating the mixture with the nuclease and the DNA polymerase; and (3)detecting the presence of cleavage of the Nucleotide with the nuclease.2. The method according to claim 1, wherein the nuclease is aribonuclease H, and the Nucleotide contains a ribonucleotide in theregion containing the base corresponding to the specific base.
 3. Themethod according to claim 1, wherein the nuclease is a restrictionenzyme, and the Nucleotide contains a recognition sequence for therestriction enzyme in the region containing the base corresponding tothe specific base.
 4. A method for detecting the presence of a basesubstitution at a specific base in a target nucleic acid, the methodcomprising: (1) mixing a sample containing a target nucleic acid with aNucleotide, wherein the Nucleotide (A) is modified at the 3′-terminussuch that extension from the terminus by a DNA polymerase does notoccur; (B) has a base sequence capable of annealing to a regioncontaining a specific base in the target nucleic acid; and (C) containsa sequence in which if there is no mismatch between the specific baseand a base corresponding to the specific base in the Nucleotide in acomplex composed of the Nucleotide and the target nucleic acid, theNucleotide is not cleaved with a nuclease, and if there is a mismatchbetween the specific base and a base corresponding to the specific basein the Nucleotide, the Nucleotide is cleaved with a nuclease to generatea new 3′-terminus; (2) treating the mixture with the nuclease and theDNA polymerase; and (3) detecting the presence of cleavage of theNucleotide with the nuclease.
 5. The method according to claim 4,wherein the nuclease is a mismatch-specific nuclease.
 6. The methodaccording to claim 1 or 4, wherein the Nucleotide has a sequence inwhich if there is no base substitution in the target nucleic acid, amismatch is not generated in the complex composed of the Nucleotide andthe target nucleic acid.
 7. The method according to claim 1 or 4,wherein the Nucleotide has a sequence in which if there is a basesubstitution in the target nucleic acid, a mismatch is not generated inthe complex composed of the Nucleotide and the target nucleic acid. 8.The method according to claim 1 or 4, wherein the cleavage of theNucleotide is detected based on the presence of an extension productgenerated by the action of the DNA polymerase.
 9. The method accordingto claim 1 or 4, wherein the cleavage of the Nucleotide is detectedbased on the presence of a fragment of a 3′ portion released from theNucleotide generated by the action of the nuclease.
 10. The methodaccording to claim 1 or 4, wherein the cleavage of the Nucleotide isdetected using a labeled compound attached to the Nucleotide.
 11. Themethod according to claim 10, wherein the labeled compound is attachedto the Nucleotide in a portion 3′ to the cleavage site for the nuclease.12. The method according to claim 10, wherein the labeled compound isattached to the Nucleotide in a portion 5′ to the cleavage site for thenuclease.
 13. The method according to claim 10, wherein the labeledcompound attached to the Nucleotide is a fluorescent substance.
 14. Themethod according to claim 13, wherein a substance capable of quenchingfluorescence is further attached to the Nucleotide, and the fluorescenceis emitted upon cleavage by the nuclease.
 15. The method according toclaim 13, wherein the cleavage of the Nucleotide is detected by afluorescence polarization method.
 16. The method according to claim 1 or4, wherein the modification of the Nucleotide at the 3′-terminus ismodification of the hydroxyl group at the 3-position of ribose.
 17. Themethod according to claim 1 or 4, wherein the Nucleotide contains anucleotide analog and/or a modified nucleotide.
 18. The method accordingto claim 17, wherein the nucleotide analog is a deoxyriboinosinenucleotide or a deoxyribouracil nucleotide, and the modifiedribonucleotide is an (α-S) ribonucleotide.
 19. The method according toclaim 1 or 4, further comprising a step of nucleic acid amplification inwhich an extension product generated by the action of the DNA polymeraseis used as a template.
 20. A method for analyzing a genotype of anallele, the method comprising detecting the presence of a basesubstitution at a specific base in a target nucleic acid according tothe method defined by claim
 19. 21. A Nucleotide used for detecting abase substitution at a specific base in a target nucleic acid, which (A)is modified at the 3′-terminus such that extension from the terminus bya DNA polymerase does not occur; (B) has a base sequence capable ofannealing to a region containing a specific base in the target nucleicacid; and (C) contains a sequence in which if there is a mismatchbetween the specific base and a base corresponding to the specific basein the Nucleotide in a complex composed of the Nucleotide and the targetnucleic acid, the Nucleotide is not cleaved with a nuclease, and ifthere is no mismatch between the specific base and a base correspondingto the specific base in the Nucleotide, the Nucleotide is cleaved with anuclease to generate a new 3′-terminus.
 22. The Nucleotide according toclaim 21, which contains a ribonucleotide in the region containing thebase corresponding to the specific base, wherein if there is no mismatchbetween the specific base and the base corresponding to the specificbase in the Nucleotide in a complex composed of the Nucleotide and thetarget nucleic acid, the Nucleotide is cleaved with a ribonuclease H.23. The Nucleotide according to claim 21, which contains a recognitionsequence for a restriction enzyme in the region containing the basecorresponding to the specific base, wherein if there is no mismatchbetween the specific base and the base corresponding to the specificbase in the Nucleotide in a complex composed of the Nucleotide and thetarget nucleic acid, the Nucleotide is cleaved with the restrictionenzyme.
 24. A Nucleotide used for detecting the presence of a basesubstitution at a specific base in a target nucleic acid, which (A) ismodified at the 3′-terminus such that extension from the terminus by aDNA polymerase does not occur; (B) has a base sequence capable ofannealing to a region containing a specific base in the target nucleicacid; and (C) contains a sequence in which if there is no mismatchbetween the specific base and a base corresponding to the specific basein the Nucleotide in a complex composed of the Nucleotide and the targetnucleic acid, the Nucleotide is not cleaved with a nuclease, and ifthere is a mismatch between the specific base and a base correspondingto the specific base in the Nucleotide, the Nucleotide is cleaved with anuclease to generate a new 3′-terminus.
 25. The Nucleotide according toclaim 24, wherein if there is a mismatch between the Nucleotide and thetarget nucleic acid in a complex composed of the Nucleotide and thetarget nucleic acid, the Nucleotide is cleaved with a mismatch-specificnuclease.
 26. The Nucleotide according to claim 21 or 24, which has asequence in which if there is no base substitution in the target nucleicacid, a mismatch is not generated in the complex composed of theNucleotide and the target nucleic acid.
 27. The Nucleotide according toclaim 21 or 24, which has a sequence in which if there is a basesubstitution in the target nucleic acid, a mismatch is not generated inthe complex composed of the Nucleotide and the target nucleic acid. 28.The Nucleotide according to claim 21 or 24, to which a labeled compoundis attached.
 29. The Nucleotide according to claim 28, wherein thelabeled compound is attached to the Nucleotide in a portion 3′ to thecleavage site for the nuclease.
 30. The Nucleotide according to claim28, wherein the labeled compound is attached to the Nucleotide in aportion 5′ to the cleavage site for the nuclease.
 31. The Nucleotideaccording to claim 28, wherein the labeled compound is a fluorescentsubstance.
 32. The Nucleotide according to claim 31, to which asubstance capable of quenching fluorescence is further attached, whereinthe fluorescence is emitted upon cleavage by the nuclease or DNAextension subsequent to the cleavage.
 33. The Nucleotide according toclaim 21 or 24, wherein the modification of the Nucleotide at the3′-terminus is modification of the hydroxyl group at the 3-position ofribose.
 34. The Nucleotide according to claim 21 or 24, which contains anucleotide analog and/or a modified nucleotide.
 35. The Nucleotideaccording to claim 34, wherein the nucleotide analog is adeoxyriboinosine nucleotide or a deoxyribouracil nucleotide, and themodified ribonucleotide is an (α-S) ribonucleotide.
 36. A kit used fordetecting a base substitution in a target nucleic acid, which containsthe Nucleotide defined by claim 21 or
 24. 37. The kit according to claim36, which contains a nuclease and/or a DNA polymerase.
 38. The kitaccording to the claim 36, which further contains a reagent fordetecting the presence of DNA extension.
 39. The kit according to claim36, which further contains a reagent for carrying out a nucleic acidamplification method.